Abstract The central aim of the proposed research is to define a novel signaling mechanism of reactive oxygen species (ROS) in mediating the interaction between cardiac myocytes and nonmyocytes which is essential for proper heart development and function, using the fruit fly Drosophila as a model system. Perturbations of the intercellular communication between cardiac myocytes and nonmyocytes are a major source of cardiac arrhythmias, sudden cardiac death and heart failure. A better understanding of the functional interactions between cardiac myocytes and nonmyocytes is therefore necessary for the development of new and improved therapies for human heart diseases. The fruit fly heart serves as an efficient model system for elucidating the precise molecular basis of cardiac myocyte-nonmyocyte functional interactions. The fly heart is a linear tube comprising of cardiomyocytes (CMs) surrounded by nonmyocyte pericardial cells (PCs). Our recently published study showed that under normal, physiological conditions, levels of ROS are elevated in the PCs compared to the CMs. ROS in PCs activate a downstream D-p38 MAPK pathway that acts on the CMs to control their development and function. These findings provide new opportunities for us to elucidate the molecular mechanisms by which ROS could mediate the functional interaction between PC and CM, which will contribute to the achieving of our central aim. Building on our published and new preliminary data, we will further delineate the components and function of the physiological ROS-D-p38 signaling pathway in the Drosophila PCs. Our central hypothesis is that a signaling pathway comprised of ROS-D-p38 (PC) ? Septate junction (SJ) proteins (PC) ? TRPA channels (CM) ? Ca2+ influx/signal transduction (CM) mediates the ROS-based paracrine interactions between PC and CM in the Drosophila heart. This hypothesis will be tested by three specific aims: (1) Determine the intracellular source(s) of ROS in pericardial cells, (2) Define the roles of SJ proteins as pericardial cell-specific targets of ROS-D-p38 signaling, and (3) Define the roles of TRPA ion channel proteins as cardiomyocyte-specific targets of ROS-D-p38 signaling. These aims will be addressed by a combination of genetic, bio-imaging and RNA-sequencing experiments. This project is expected to reveal novel and important insights into the poorly understood areas of ROS-based paracrine signaling and functional interactions between cardiac myocytes and nonmyocytes. This knowledge may facilitate the development of new and improved therapies for human heart disease and failure.